Abstract: A novel water-soluble heteropolysaccharide (PEPS1), with a molecular weight of 1.88×104 Da as determined by high performance liquid chromatography (HPLC), specifically size exclusion chromatography, was isolated from the fruiting bodies of Pleurotus eryngii by hot water extraction and further purification by DEAE Sepharose Fast Flow and Sephacryl S-300 and S-100 High-Resolution chromatography. By use of compositional analysis, methylation analysis, together with 1H, 13C NMR and 2D NMR spectroscopy including COSY, TOCSY, HMQC, HMBC and NOESY experiments, the PEPS1 was identified as a heteropolysaccharide that consists of a α-d-(1→6)-Galactopyranan backbone with a β-d-Mannosyl unit on O-2 of the 2,6-di-O-substituted-d-Galactosyl units, α-(1→6)-3-O-Me-d-Galactopyranan backbone with a terminal α-3-O-Me-d-Galactosyl unit and it also contained a minor 2,3,6-α-d-Galatopyranan units and 4-β-d-Mannopyranan residues. [Copyright &y& Elsevier]